The industrial applicability of microorganisms is restricted by their physiological limits set by solvent, pH, various solutes, salts and temperature. Organic solvents are generally toxic to microorganisms even at low concentrations. The toxicity of solvents significantly limits use of microorganisms in industrial biotechnology for production of specialty chemicals and for bioremediation applications. Solvent molecules incorporate into bacterial membranes and disrupt membrane structure (Isken and Bont, 1998, Extremophiles 2(3): 229–238); (Pinkart and White, 1997, J. Bacteriol. 179(13): 4219–4226); (Ramos, Duque et al., 1997, “J. Biol. Chem. 272(7): 3887–3890); (Alexandre, Rousseaux et al., 1994, FEMS Microbiol, Lett, 124(1): 17–22); and Kieboom, Dennis et al., 1998, J. of Bacteriology 180(24): 6769–6772). Classic strain improvement methods including UV and chemical mutagenesis have been applied for selection of more tolerant strains (Miller, J., “A Short Course In Bacterial Genetics,” Cold Spring Harbor Laboratory Press, 1992). A number of studies have been dedicated to identification and isolation of solvent tolerant mutants among various bacterial strains. Spontaneous E. coli solvent tolerant mutants and mutants isolated in the process of 1-methyl-3-nitrosoguanidine (NTG) mutagenesis were obtained from strain K-12 (Aono, Aibe et al., 1991 Agric. Biol. Chem 55(7): 1935–1938). The mutants could grow in the presence of diphenylether, n-hexane, propylbenzene, cyclohexane, n-pentane, p-xylene. Various Pseudomonas strains were able to adapt and to grow in a toluene-water two-phase system (Inoue and Horikoshi, 1989, Nature 338: 264–266), with p-xylene (Cruden, Wolfram et al., 1992, Appl. Environ. Microbiol. 58(9): 2723–2729), styrene and other organic solvents (Weber, Ooijkaas et al., 1993, Appl. Environ. Microbiol. 59(10): 3502–3504), (de Bont 1998, Trends in Biotechnology 16: 493–499). Yomano et al. isolated ethanol tolerant mutants which increased tolerance from 35 g/l to 50 g/l during 32 consequent transfers (Yomano, York et al., 1998, J. Ind. Microbiol. Biotechnol. 20(2): 132–138). High temperature evolution using E. coli has been disclosed in the art (Bennett, 1990, Nature, Vol. 346, 79–81) however the fitness gain was low as compared to the parent.
Strains of E. coli that carry mutations in one of the DNA repair pathways have been described which have a higher random mutation rate than that of typical wild type strains (see, Miller supra, pp. 193–211). As reported by Degenen and Cox (J. Bacteriol., 1974, Vol. 117, No. 2, pp. 477–487), an E. coli strain carrying a mutD5 allele demonstrates from 100 to 10,000 times the mutation rate of its wild type parent. Greener et al., “Strategies In Molecular Biology,” 1994, Vol. 7, pp. 32–34, disclosed a mutator strain that produces on average one mutation per 2000 bp after growth for about 30 doublings.
Microorganisms are used industrially to produce desired proteins, such as hormones, growth factors and enzymes and to produce chemicals, such as glycerol and 1,3 propanediol (WO 98/21340 published May 22, 1998 and U.S. Pat. No. 5,686,276 issued Nov. 11, 1997), vitamins, such as ascorbic acid intermediates (1985, Science 230:144–149), amino acids, and dyes, such as indigo (U.S. Pat. No. 4,520,103, issued May 28, 1985). In spite of advances in the art, there remains a need to improve the microorganisms and methods for producing such desired proteins, chemicals, amino acids and dyes.